Composite Bone Graft Substitute Cement and Articles Produced Therefrom

ABSTRACT

The invention provides a particulate composition adapted for forming a bone graft substitute cement upon mixing with an aqueous solution, including i) a calcium sulfate hemihydrate powder having a bimodal particle distribution and a median particle size of about 5 to about 20 microns, wherein the calcium sulfate hemihydrate is present at a concentration of at least about 70 weight percent based on the total weight of the particulate composition; ii) a monocalcium phosphate monohydrate powder; and iii) a β-tricalcium phosphate powder having a median particle size of less than about 20 microns. Bone graft substitute cements made therefrom, a bone graft substitute kit comprising the particulate composition, methods of making and using the particulate composition, and articles made from the bone graft substitute cement are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a continuation of application Ser. No. 11/530,085,filed Sep. 8, 2006, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/715,542, filed Sep. 9, 2005, and both priorapplications are incorporated herein by reference in their entirety andfor all purposes.

FIELD OF THE INVENTION

The invention is directed to a particulate composition adapted forforming a bone graft substitute cement upon mixing with an aqueoussolution, a bone graft substitute cement made therefrom, a bone graftsubstitute kit comprising the particulate composition, methods of makingand using the particulate composition, and articles made from the bonegraft substitute cement.

BACKGROUND OF THE INVENTION

Defects in bone structure arise in a variety of circumstances, such astrauma, disease, and surgery. There is a need for effective repair ofbone defects in various surgical fields, including maxillo-craniofacial,periodontics, and orthopedics. Numerous natural and synthetic materialsand compositions have been used to stimulate healing at the site of abone defect. As with compositions used to repair other types of tissue,the biological and mechanical properties of a bone repair material arecritical in determining the effectiveness and suitability of thematerial in any particular application.

After blood, bone is the second most commonly transplanted material.Autologous cancellous bone has long been considered the most effectivebone repair material, since it is both osteoinductive andnon-immunogenic. However, adequate quantities of autologous cancellousbone are not available under all circumstances, and donor site morbidityand trauma are serious drawbacks to this approach. The use of allograftbone avoids the problem of creating a second surgical site in thepatient, but suffers from some disadvantages of its own. For instance,allograft bone typically has a lower osteogenic capacity than autograftbone, a higher resorption rate, creates less revascularization at thesite of the bone defect, and typically results in a greater immunogenicresponse. The transfer of certain diseases is also a danger when usingallografts.

To avoid the problems associated with autograft and allograft bone,considerable research has been conducted in the area of synthetic bonesubstitute materials that can be used in lieu of natural bone. Forexample, various compositions and materials comprising demineralizedbone matrix, calcium phosphate, and calcium sulfate have been proposed.

Cements comprising calcium sulfate have a long history of use as bonegraft substitutes. Modern surgical grade calcium sulfate cements offerhigh initial strength, good handling properties, and are consistentlyreplaced by bone in many applications. However, calcium sulfate cementsare characterized by relatively rapid resorption by the body, which canbe undesirable in certain applications.

Hydroxyapatite is one of the most commonly used calcium phosphates inbone graft materials. Its structure is similar to the mineral phase ofbone and it exhibits excellent biocompatibility. However, hydroxyapatitehas an extremely slow resorption rate that may be unsuitable in certainapplications. Other calcium phosphate materials have also been used inthe art, such as β-tricalcium phosphate, which exhibits a fasterresorption rate than hydroxyapatite, but has less mechanical strength.Certain calcium phosphate materials that set in situ have also beenattempted, such as mixtures of tetracalcium phosphate and dicalciumphosphate anhydrate or dihydrate, which react to form hydroxyapatitewhen mixed with an aqueous solution.

The presently available synthetic bone repair materials do not presentideal functional characteristics for all bone graft applications. Asnoted above, some compositions exhibit a resorption rate that is eithertoo slow or too rapid. Further, many bone graft cements are difficult toimplant because they fail to set or cannot be injected. Other drawbacksare inadequate strength and difficulty in adding biologically activesubstances for controlled release. For these reasons, there remains aneed in the art for bone graft cement compositions that combine adesirable resorption rate with high mechanical strength, ease ofhandling, and osteoconductivity.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a particulate composition adapted forforming a bone graft substitute cement upon mixing with an aqueoussolution, as well as a hardened bone graft substitute cement madetherefrom. The invention also relates to kits comprising the particulatecomposition, and methods of making and using the composition. Theparticulate composition of the invention comprises a calcium sulfatehemihydrate powder in combination with a brushite-forming calciumphosphate mixture. Upon mixing the particulate composition with anaqueous mixing solution, a hardened biphasic cement comprising brushiteand calcium sulfate dihydrate is formed. The calcium sulfate dihydrateprovides good mechanical strength and, due to its relatively fastresorption rate, is rapidly replaced with bone tissue in the resultingcement, while the brushite serves to reduce the overall resorption rateof the cement as compared to a cement composition solely comprisingcalcium sulfate dihydrate. Certain embodiments of the bone substitutecement of the invention exhibit high mechanical strength, such as highcompressive strength and diametral tensile strength, set into a hardenedcomposition within a reasonable period of time, facilitate developmentof high quality bone at the site of the bone defect, and exhibitacceptable handling characteristics.

In one aspect, the invention provides a particulate compositioncomprising a mixture of a calcium sulfate hemihydrate powder having abimodal particle distribution and a median particle size of about 5 toabout 20 microns, and a brushite-forming calcium phosphate composition.The brushite-forming calcium phosphate mixture comprises monocalciumphosphate monohydrate powder and a β-tricalcium phosphate powder. Theβ-tricalcium phosphate powder has a median particle size of less thanabout 20 microns. The calcium sulfate hemihydrate powder is present at aconcentration of at least about 50 weight percent based on the totalweight of the particulate composition, more preferably at least about 70weight percent, and most preferably at least about 75 weight percent.The brushite-forming calcium phosphate composition is typically presentat a concentration of about 3 to about 30 weight percent based on thetotal weight of the particulate composition.

The β-tricalcium phosphate powder portion of the particulate compositionpreferably has a bimodal particle size distribution characterized byabout 30 to about 70 volume percent of particles having a mode of about2.0 to about 6.0 microns and about 30 to about 70 volume percent ofparticles having a mode of about 40 to about 70 microns based on thetotal volume of the β-tricalcium phosphate powder. In anotherembodiment, the bimodal particle size distribution comprises about 50 toabout 65 volume percent of particles having a mode of about 4.0 to about5.5 microns and about 35 to about 50 volume percent of particles havinga mode of about 60 to about 70 microns based on the total volume of theβ-tricalcium phosphate powder.

The calcium sulfate hemihydrate portion of the particulate compositionpreferably comprises α-calcium sulfate hemihydrate, and the bimodalparticle distribution preferably comprises about 30 to about 60 volumepercent of particles having a mode of about 1.0 to about 3.0 microns,and about 40 to about 70 volume percent of particles having a mode ofabout 20 to about 30 microns, based on the total volume of the calciumsulfate hemihydrate powder.

The particulate composition mixture may further comprise β-tricalciumphosphate granules having a median particle size of at least about 75microns, such as about 75 to about 1,000 microns. The β-tricalciumphosphate granules are typically present at a concentration of up toabout 20 weight percent based on the total weight of the particulatecomposition, and more preferably at a concentration of up to about 12weight percent.

The particulate composition may comprise further additives, such as anaccelerant adapted for accelerating the conversion of calcium sulfatehemihydrate to calcium sulfate dihydrate. An example of such anaccelerant is sucrose-coated calcium sulfate dihydrate particles.Further, the composition may comprise a biologically active agent, suchas cancellous bone chips, growth factors, antibiotics, pesticides,chemotherapeutic agents, antivirals, analgesics, anti-inflammatoryagents, and osteoinductive or osteoconductive materials. Demineralizedbone matrix is one preferred biologically active agent.

In one embodiment, the particulate composition of the invention sets toa hardened mass upon mixing with an aqueous solution in about 3 to about25 minutes. Thus, in another aspect of the invention, a bone graftsubstitute cement is provided, the cement comprising the paste formed bymixing the particulate composition of the invention with an aqueoussolution. The bone graft substitute cement can comprise β-tricalciumphosphate granules (if present) and a reaction product formed by mixinga particulate composition of the invention with an aqueous solution, thereaction product comprising calcium sulfate dihydrate and brushite. Thebone graft substitute cement can be cast in a predetermined shape, suchas pellets, granules, wedges, blocks, and disks, molded into a desiredshape at the time of application, or simply injected or otherwisedelivered to the site of a bone defect without prior molding or shaping.The cement of the invention can also be incorporated into any of variousorthopedic implant devices, typically being applied in the form of outercoatings or as filling material in porous outer layers of such devicesin order to facilitate bone ingrowth in the area of the implanteddevice.

The hardened bone graft substitute cement preferably exhibits certainmechanical strength characteristics, such as a diametral tensilestrength of at least about 4 MPa after curing for one hour in ambientair following mixing of the particulate composition with an aqueoussolution, more preferably a diametral tensile strength of at least about5 MPa, most preferably at least about 6 MPa. Further, preferredembodiments of the bone graft substitute cement exhibit a diametraltensile strength of at least about 8 MPa after curing for 24 hours inambient air following mixing of the particulate composition with anaqueous solution, more preferably a diametral tensile strength of atleast about 9 MPa after curing for 24 hours, and most preferably atleast about 10 MPa.

Preferred embodiments of the bone graft substitute cement also exhibit ahigh level of compressive strength, such as a compressive strength of atleast about 15 MPa after curing for one hour in ambient air followingmixing of the particulate composition with an aqueous solution, morepreferably a compressive strength of at least about 40 MPa. Further,preferred embodiments of the bone graft substitute cement will exhibit acompressive strength of at least about 50 MPa after curing for 24 hoursin ambient air following mixing of the particulate composition with anaqueous solution, more preferably a compressive strength of at leastabout 80 MPa.

Preferred embodiments of the bone graft substitute cement also exhibitan average dissolution rate, expressed as an average percentage ofweight loss per day, that is at least about 25% lower than the averagedissolution rate of a cement formed using a particulate compositionconsisting of calcium sulfate, the average dissolution rate measured byimmersion of a 4.8 mm OD pellet having a length of 3.3 mm in distilledwater at 37° C. More preferably, the average dissolution rate is atleast about 30% lower or at least about 35% lower.

In yet another aspect, the present invention provides a bone graftsubstitute kit, comprising at least one container enclosing theparticulate composition according to the invention, a separate containerenclosing a sterile aqueous solution, and a written instruction setdescribing a method of using the kit. The bone graft substitute kit mayfurther comprise a mixing apparatus for mixing the aqueous solution withthe particulate composition, and a device for delivering the bone graftsubstitute cement to the site of a bone defect, such as an injectiondevice (e.g., a syringe).

In a further aspect of the invention, a method for treating a bonedefect is provided. The method comprising applying the above-describedbone graft substitute cement to the site of the bone defect. As notedabove, the bone graft substitute cement can be administered in the formof a precast molded form, molded immediately prior to administrationinto the desired shaped based on the size and shape of the bone defect,or administered using an injection device or other means of deliveringthe composition directly to the bone defect without prior molding.

In a still further aspect of the invention, a method of forming theparticulate composition of the invention is provided. The methodtypically comprises mixing or blending each powder or granule componentof the particulate composition in order to form a homogenous mixture.Thus, in one embodiment, the method of forming the particulatecomposition comprises mixing the β-tricalcium phosphate powder, thecalcium sulfate hemihydrate powder (which can be optionally acceleratedby the addition of an accelerant as noted above), monocalcium phosphatemonohydrate powder, and β-tricalcium phosphate granules (if present).The mixing of the various powder or granular ingredients preferablyoccurs immediately prior to mixing of the particulate composition withthe aqueous solution.

The aqueous solution mixed with the particulate composition in order toform the setting cement preferably comprises sterile water, and mayinclude at least one carboxylic acid therein. For example, thecarboxylic acid can be glycolic acid or other hydroxy carboxylic acids.Preferably, the acid is neutralized to a neutral pH of approximately6.5-7.5.

In another aspect of the invention, methods, compositions, and kits areprovided for enhancing the storage stability of the components of thebone graft substitute composition of the invention. In one embodiment,the brushite-forming calcium phosphate materials (i.e., β-tricalciumphosphate powder and monocalcium phosphate monohydrate powder) areeither stored separately prior to preparation of the bone graftsubstitute cement (e.g., placed in separate containers in a kit) orhermetically packaged in a completely dry environment in order toprevent reaction of the two calcium phosphate compounds. In anotherembodiment, the organic carboxylic acid component discussed above inconnection with the aqueous mixing solution is packaged as a crystallinepowder (e.g., in neutralized salt form such as an alkali metal salt)with the remaining particulate components of the kit rather than insolution. Using the acid component in powder form avoids degradation ofthe acid upon sterilization of the composition with gamma radiation,which can lead to undesirable increases in the setting time of the bonegraft substitute cement of the invention.

Thus, in one embodiment, the invention provides a method for improvingthe storage stability of a kit comprising a particulate composition andan aqueous solution adapted for forming a bone graft substitute cementupon mixing, wherein the kit includes calcium phosphate powders reactiveto form brushite in the presence of water and a carboxylic acid, themethod comprising: i) packaging a monocalcium phosphate monohydratepowder and a β-tricalcium phosphate powder in separate containers in thekit; and ii) packaging the carboxylic acid in the kit either in the formof a crystalline powder or dissolved in the aqueous solution, with theproviso that when the carboxylic acid is dissolved in the aqueoussolution, it is added to the solution after radiation sterilization ofthe aqueous solution. The kit may further comprise calcium sulfatehemihydrate powder, and the method may further comprise packaging thecalcium sulfate hemihydrate powder in a separate container, or inadmixture with one or both of the monocalcium phosphate monohydratepowder and the β-tricalcium phosphate powder. The method will typicallyfurther comprise irradiating the components of the kit with gammaradiation for sterilization.

Exemplary neutralized salts of carboxylic acids that can be utilized asthe carboxylic acid powder include sodium glycolate, potassiumglycolate, sodium lactate, and potassium lactate. The carboxylic acidcrystalline powder is typically packaged separately in a container orpackaged in the container containing the monocalcium phosphatemonohydrate powder or in the container containing the β-tricalciumphosphate powder.

In another embodiment of the invention, a bone graft substitute kit isprovided, comprising: i) a first container enclosing a monocalciumphosphate monohydrate powder; ii) a second container enclosing aβ-tricalcium phosphate powder; iii) a calcium sulfate hemihydrate powderenclosed within a separate container or admixed with one or both of themonocalcium phosphate monohydrate powder and the β-tricalcium phosphatepowder; iv) an aqueous solution enclosed within a separate container;and v) a carboxylic acid dissolved within the aqueous solution orpresent in the form of a crystalline powder, the carboxylic acidcrystalline powder being enclosed within a separate container or admixedwith any one or more of the monocalcium phosphate monohydrate powder,the β-tricalcium phosphate powder, and the calcium sulfate hemihydratepowder, with the proviso that when the carboxylic acid is dissolved inthe aqueous solution, it is added to the solution after radiationsterilization of the aqueous solution. In certain embodiments, thecarboxylic acid crystalline powder is enclosed within a separatecontainer such that the carboxylic acid crystalline powder can bereconstituted by admixture with the aqueous solution prior to mixing theaqueous solution with one or more of the monocalcium phosphatemonohydrate powder, the β-tricalcium phosphate powder, and the calciumsulfate hemihydrate powder.

The calcium sulfate hemihydrate powder may further include, inadmixture, an accelerant adapted for accelerating the conversion ofcalcium sulfate hemihydrate to calcium sulfate dihydrate. Additionally,the kit may further include β-tricalcium phosphate granules in aseparate container or in admixture with one or more of the monocalciumphosphate monohydrate powder, the β-tricalcium phosphate powder, and thecalcium sulfate hemihydrate powder. A biologically active agent can alsobe included in the kit and enclosed within a separate container oradmixed with any one or more of the monocalcium phosphate monohydratepowder, the β-tricalcium phosphate powder, and the calcium sulfatehemihydrate powder.

In yet another embodiment, a bone graft substitute kit is provided,comprising:

i) a first container enclosing a monocalcium phosphate monohydratepowder; ii) a second container enclosing a β-tricalcium phosphate powderhaving a median particle size of less than about 20 microns; iii) anα-calcium sulfate hemihydrate powder enclosed within a separatecontainer or admixed with the β-tricalcium phosphate powder in thesecond container, the α-calcium sulfate hemihydrate powder having abimodal particle distribution and a median particle size of about 5 toabout 20 microns; iv) an aqueous solution enclosed within a separatecontainer; v) a carboxylic acid in the form of a crystalline powder, thecarboxylic acid crystalline powder being enclosed within a separatecontainer, wherein the carboxylic acid is in the form of a neutralizedalkali metal salt; vi) an accelerant adapted for accelerating theconversion of calcium sulfate hemihydrate to calcium sulfate dihydratein admixture with the α-calcium sulfate hemihydrate powder; and vii)β-tricalcium phosphate granules in a separate container or in admixturewith one or both of the β-tricalcium phosphate powder and the calciumsulfate hemihydrate powder, wherein the granules have a median particlesize of at least about 75 microns.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, wherein:

FIG. 1 graphically illustrates the concept of a bimodal particle sizedistribution plot based on high resolution laser diffraction;

FIGS. 2 a, 2 b, and 2 c provide several views of an exemplary diametraltensile strength specimen mold;

FIG. 3 graphically illustrates a comparison of diametral tensilestrength of a bone graft cement according to the invention and acommercially available calcium sulfate cement;

FIG. 4 graphically illustrates the in vitro dissolution properties oftwo bone graft cements according to the invention as compared to acommercially available calcium sulfate cement; and

FIG. 5 graphically illustrates titration curves for solutions made usingnon-irradiated and gamma irradiated crystalline glycolic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings. The invention may be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will satisfy applicable legal requirements. As usedin this specification and the claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

The present invention provides a particulate composition useful as abone graft substitute cement that hardens or sets upon mixing with anaqueous solution. The particulate composition includes a calcium sulfatehemihydrate (hereinafter “CSH”) powder and a brushite-forming calciumphosphate mixture comprising monocalcium phosphate monohydrate(hereinafter “MCPM”) powder and a β-tricalcium phosphate (hereinafter“β-TCP”) powder.

Use of the particulate composition of the invention produces a bonegraft substitute cement comprising calcium sulfate dihydrate(hereinafter “CSD”), which is the product of the reaction between CSHand water. The CSD component of the cement confers good mechanicalstrength to the cement, stimulates bone growth, and provides arelatively fast resorption rate in vivo, such that a porous structure inthe cement is quickly created upon implantation. Thus, the CSD componentof the cement can be rapidly replaced with bone tissue ingrowth into theimplant site.

The two calcium phosphate components react to form brushite upon mixingwith an aqueous solution. The presence of the brushite in the cementslows the resorption rate of the bone graft substitute cement ascompared to a cement comprising CSD only. Thus, the biphasic bone graftsubstitute cement of the invention provides a dual resorption ratedefined by the CSD component and the brushite component.

In addition to a slower resorption rate, embodiments of the particulatecomposition of the invention can provide a bone graft substitute cementthat exhibits high mechanical strength, good handling characteristics,and a reasonable setting time. Additionally, certain embodiments of thebone graft substitute cement of the invention are capable of producinghigh quality bone when used to treat bone defects.

The CSH powder used in the present invention preferably has a bimodalparticle distribution. As understood in the art, a bimodal particledistribution refers to a particle distribution characterized by twopeaks in a plot of particle size vs. the volume percentage of particlesof each size. FIG. 1 illustrates an exemplary bimodal particle sizedistribution plot. In a preferred embodiment, the bimodal particledistribution of the CSH powder is characterized by about 30 to about 60volume percent of particles having a mode of about 1.0 to about 3.0microns and about 40 to about 70 volume percent of particles having amode of about 20 to about 30 microns, based on the total volume of theCSH powder. In yet another embodiment, the bimodal particle distributioncomprises about 40 to about 60 volume percent of particles having a modeof about 1.0 to about 2.0 microns and about 40 to about 60 volumepercent of particles having a mode of about 20 to about 25 microns. Themedian particle size of the CSH powder is preferably about 5 to about 20microns, more preferably about 8 to about 15 microns, and mostpreferably about 10 to about 15 microns.

As used herein, “median particle size” refers to the particle size thatdivides a population of particles in half such that half of the volumeof particles in the population is above the median size and half isbelow. Median particle size is measured using linear interpolation ofdata acquired through a high resolution laser diffraction method. Morespecifically, the laser diffraction method is performed with parallellight with a constant frequency of 632.8 nanometers and which exhibits 5milliwatts of power. Measurements of laser diffraction are acquiredthrough a 32 channel detector array. Particle delivery to measurementsystem is performed through a relatively constant mass flow rate usingan optimum dispersing media such as air flow creating a −3.5 bar gaugepressure. A commercially available machine for laser-diffractionparticle analysis is the OASIS (Sympatec; Clausthal-Zellerfeld, Germany)dispersing unit. The OASIS system is used in the dry mode via the VIBRImodel HDD200 and RODOS M. The VIBRI model is used with a 75% feed rateand 3.0 mm gap. The −3.5 bar gauge pressure is produced through a 4 mminjector. For measuring particle size of calcium sulfate hemihydrate,the R2 lens (0.25/0.45 . . . 87.5 um) is preferred, and for tricalciumphosphate components, the R4 lens (0.5/1.8 . . . 350 um) is preferred(both also from Sympatec).

The particulate composition in the invention preferably comprises a CSHpowder in an amount of at least about 50 weight percent based on thetotal weight of the particulate composition, more preferably at leastabout 70 weight percent, and most preferably at least about 75 weightpercent. In certain embodiments, the CSH powder is present in an amountof at least about 80 weight percent, at least about 85 weight percent,or at least about 90 weight percent. Typically, the CSH powder ispresent in an amount of about 70 weight percent to about 99 weightpercent, more preferably about 70 weight percent to about 90 weightpercent.

The CSH is preferably α-calcium sulfate hemihydrate, which exhibitshigher mechanical strength as compared to the beta form upon setting toform CSD. The CSH portion of the particulate composition is importantfor providing mechanical strength to the resulting bone graft substitutecement, as well as contributing to the ability to set or harden in arelatively short period of time. As is known in the art, CSH has theformula CaSO₄.½H₂O, and will react with water to form calcium sulfatedihydrate (CaSO₄.2H₂O). It is believed that the presence of CSD in thebone graft substitute cement of the invention contributes to rapidregeneration of bone tissue at the site of the bone defect.

CSH powder can be formed by dehydration of the dihydrate form byheating. Depending on the method of heating, the alpha or beta form isobtained. The two forms exhibit crystallographic and particle morphologydifferences. The preferred alpha form, which has a higher density, istypically characterized by large, hexagonal shaped rod-like primarycrystals that are compact and well formed with sharp edges.

In a preferred embodiment, the CSH powder is made by the processdisclosed in U.S. Pat. No. 2,616,789, which is incorporated entirelyherein by reference in its entirety. The process involves immersion ofcalcium sulfate dihydrate in a solution of water and an inorganic salt.Preferred salts include magnesium chloride, calcium chloride, and sodiumchloride. However, other inorganic salts can be used without departingfrom the invention, such as ammonium chloride, ammonium bromide,ammonium iodide, ammonium nitrate, ammonium sulfate, calcium bromide,calcium iodide, calcium nitrate, magnesium bromide, magnesium iodide,magnesium nitrate, sodium bromide, sodium iodide, sodium nitrate,potassium chloride, potassium bromide, potassium iodide, potassiumnitrate, cesium chloride, cesium nitrate, cesium sulfate, zinc chloride,zinc bromide, zinc iodide, zinc nitrate, zinc sulfate, cupric chloride,cupric bromide, cupric nitrate, cupric sulfate, and mixtures thereof.Preferred salts are biocompatible, and any of the salts can be used intheir anhydrous or hydrate forms. Reference to the salt is intended toencompass both anhydrous and hydrate forms. The calcium sulfatedihydrate and the solution are heated to substantially the boiling pointat atmospheric pressure until a substantial portion of the calciumsulfate dihydrate is converted to CSH. The resulting CSH has a differentcrystalline structure than CSH produced by other hydrothermal processesand has a lower water-carrying capacity after being milled. Inparticular, the crystalline structure of the CSH made according to thismethod is characterized by thick, stubby, rod-like crystals.

In one embodiment, the CSH powder further includes an accelerant capableof accelerating the conversion of CSH to the dihydrate form, therebycausing the bone graft substitute cement made therefrom to set morequickly. Although not wishing to be bound by a theory of operation, itis believed that the accelerant particles act as crystallizationnucleation sites for the conversion of CSH to calcium sulfate dihydrate.Examples of accelerants include calcium sulfate dihydrate, potassiumsulfate, sodium sulfate, or other ionic salts. A preferred accelerant iscalcium sulfate dihydrate crystals (available from U.S. Gypsum) coatedwith sucrose (available from VWR Scientific Products). A process ofstabilizing the dihydrate crystals by coating with sucrose is describedin U.S. Pat. No. 3,573,947, which is hereby incorporated by reference inits entirety. The accelerant is typically present in an amount of up toabout 1.0 weight percent, based on the total weight of the particulatecomposition. In some embodiments, the particulate composition includesbetween about 0.001 and about 0.5 weight percent of the accelerant, moretypically between about 0.01 and about 0.3 weight percent. Mixtures oftwo or more accelerants can be used.

The calcium phosphate portion of the particulate composition of theinvention comprises a MCPM powder (Ca(H₂PO₄)₂—H₂O) and a β-TCP powder(Ca₃(PO₄)₂). As understood in the art, the main reaction product of MCPMand β-TCP is brushite, otherwise known as dicalcium phosphate dihydrate(CaHPO₄.2H₂O) (DCPD). The brushite-forming powders may also participatein other reactions that would result in the formation of certain calciumphosphates with a greater thermodynamic stability than DCPD, such ashydroxyapatite, octacalcium phosphate, and the like. A certain amount ofthe β-TCP powder may also remain unreacted in the cement.

The β-TCP powder preferably has a median particle size of less thanabout 20 microns, and more preferably a median particle size of lessthan about 18 microns, and most preferably a median particle size ofless than about 15 microns. Typically the β-TCP powder will have amedian particle size of about 10 microns to about 20 microns. The sizeof the β-TCP powder may affect the amount of brushite formed in the bonegraft substitute cement. It is believed that smaller particle sizes ofβ-TCP will result in an increased rate of brushite formation, and largerparticle sizes will result in a lower rate of brushite formation. It istypically preferred to use smaller β-TCP particles in order to increasethe brushite-forming reaction rate.

The β-TCP powder portion of the particulate composition preferably has abimodal particle size distribution characterized by about 30 to about 70volume percent of particles having a mode of about 2.0 to about 6.0microns and about 30 to about 70 volume percent of particles having amode of about 40 to about 70 microns based on the total volume of theβ-tricalcium phosphate powder. In one embodiment, the β-TCP powder has abimodal particle size distribution characterized by about 50 to about 65volume percent of particles having a mode of about 4.0 to about 5.5microns and about 35 to about 50 volume percent of particles having amode of about 60 to about 70 microns based on the total volume of theβ-tricalcium phosphate powder.

The MCPM powder is relatively soluble in water, which means particlesize is relatively unimportant. Typically, the MCPM powder will have aparticle size of less than about 350 microns; however, other particlessize could be utilized without departing from the invention. As would beunderstood, MCPM is the hydrate form of monocalcium phosphate (MCP). Asused herein, reference to MCPM is intended to encompass MCP, which issimply the anhydrous form of MCPM that releases the same number ofcalcium and phosphoric acid ions in solution. However, if MCP is used inplace of MCPM, the amount of water used to form the bone graftsubstitute cement would need to be increased to account for the watermolecule missing from MCP (if it is desired to produce precisely thesame dissolution product as formed when using MCPM).

As noted above, the brushite component of the bone graft substitutecement of the invention serves to slow the in vivo resorption of thebone graft substitute cement as compared to a calcium sulfate cement. Inturn, the slower resorption rate may enable the bone graft substitutecement to provide structural support at the site of the bone defect forlonger periods of time, which can aid the healing process in certainapplications. Although not bound by any particular theory of operation,it is believed that the bone graft substitute cement of the inventionwill become a highly porous matrix of calcium phosphate material afterbeing administered in vivo due to the relatively quick resorption of thecalcium sulfate component of the mixture. The remaining porous matrix ofcalcium phosphate provides excellent scaffolding for bone ingrowthduring the natural healing process.

The amount of MCPM powder and β-TCP powder present in the particulatecomposition can vary and depends primarily on the amount of brushitedesired in the bone graft substitute cement. The brushite-formingcalcium phosphate composition (i.e., the combined amount of MCPM andβ-TCP powders) will typically be present at a concentration of about 3to about 30 weight percent based on the total weight of the particulatecomposition, more preferably about 10 to about 20 weight percent, mostpreferably about 15 weight percent. The relative amounts of MCPM andβ-TCP can be selected based on their equimolar, stoichiometricrelationship in the brushite-forming reaction. In one embodiment, theMCPM powder is present at a concentration of about 3 to about 7 weightpercent, based on the total weight of the particulate composition, andthe β-TCP is present in an amount of about 3.72 to about 8.67 weightpercent.

It has been discovered that the MCPM and β-TCP powders can reactprematurely during storage in the presence of residual moisture to formbrushite and/or monetite, an undesirable anhydrous analog of brushite.Thus, storage of the brushite-forming calcium phosphate powders togetherin a homogenous mixture can result in reduction in the amount ofbrushite produced upon mixing the particulate composition with theaqueous mixing solution to form the bone graft substitute cement, whichin turn, can alter the properties of the bone graft substitute cement inan undesirable manner. As a result, in a preferred embodiment, the twocalcium phosphate components are either packaged together in a dryenvironment and hermetically sealed against moisture invasion duringstorage or are packaged separately during storage. In one embodiment,the two calcium phosphate powders are packaged separately, wherein eachpowder is either packaged alone with no other components of theparticulate composition of the invention or in admixture with one ormore of the remaining components (e.g., the CSH powder).

In certain embodiments, the particulate composition of the inventionwill also include a plurality of β-TCP granules having a median particlesize greater than the median particle size of the β-TCP powder. Theβ-TCP granules typically have a median particle size of about 75 toabout 1,000 microns, more preferably about 100 to about 400 microns, andmost preferably about 180 to about 240 microns. The granules serve tofurther reduce the resorption rate of the bone graft substitute cementand contribute to scaffold formation. The β-TCP granules are typicallypresent at a concentration of up to about 20 weight percent, based onthe total weight of the particulate composition, more preferably up toabout 15 weight percent based on the total weight of the composition,and most preferably up to about 12 weight percent. In one preferredembodiment, the β-TCP granules are present at a concentration of about 8to about 12 weight percent. The β-TCP granules can provide a relativelyinert third phase in the final cement that exhibits an even slowerresorption rate than the brushite formed by reaction of the MCPM and theβ-TCP powder. Thus, the presence of the granules can further alter theresorption profile of the resulting bone graft substitute cement.

Both the β-TCP granules and the β-TCP powder used in the presentinvention can be formed using a commercially available β-TCP powder as astarting material, such as β-TCP powder available from Plasma BiotalLtd. (Derbyshire, UK). In one embodiment, the β-TCP components of theparticulate composition are formed by first wet milling a commerciallyavailable β-TCP powder in a ball mill to a median particle size of lessthan 1.0 micron and then draining the resulting slurry through astrainer to remove the milling media. Thereafter, the solid cake ofβ-TCP can be separated from any remaining liquid components using any ofa variety of techniques known in the art, such as centrifuging, gravityseparation, filter pressing, evaporation, and the like. The dry cake isthen processed through a series of sieves in order to produce twoseparate β-TCP components having different median particle sizes. Thedried cake of β-TCP is typically milled either during or prior tosieving in order to fragment the cake. In one preferred embodiment, thesystem of sieves produces a β-TCP component having a particle size rangeof about 125 to about 355 microns in a green (i.e., unfired) state andanother β-TCP component having a particle size range of about 75 toabout 355 microns in a green state. Thereafter, the two β-TCP componentsare sintered, and thereby densified, by heat treatment in a furnace. Inone embodiment, the furnace treatment involves heating the β-TCP powdercomponents on an alumina plate at a temperature of about 1100-1200° C.for about three hours. It is typical to ramp the temperature up to thedesired sintering temperature and ramp the temperature back down duringthe cooling period at a rate no greater than about 5-6° C. per minute.

Following the sintering process, the densified β-TCP granules having hada green state particle size of about 125 to about 355 microns can beused as the granule component of the particulate composition. Thesintered β-TCP component having had a green (i.e., unfired) stateparticle size of about 75 to about 355 microns can be dry milled in aball mill for approximately one to four hours in order to form the β-TCPpowder having a median particle size of less than about 20 microns,which can then be used in the particulate composition as describedabove.

The aqueous component that is mixed with the particulate composition ofthe invention is selected in order to provide the composition with adesired consistency and hardening or setting time. Typically, theaqueous solution is provided in an amount necessary to achieve a liquidto powder mass ratio (L/P) of at least about 0.2, more preferably atleast about 0.21, and most preferably at least about 0.23. A preferredL/P ratio range is about 0.2 to about 0.3, more preferably about 0.2 toabout 0.25.

Examples of suitable aqueous components include water (e.g., sterilewater) and solutions thereof, optionally including one or more additivesselected from the group consisting of sodium chloride, potassiumchloride, sodium sulfate, potassium sulfate, EDTA, ammonium sulfate,ammonium acetate, and sodium acetate. In one preferred embodiment, theaqueous mixing solution used is a saline solution or a phosphatebuffered saline solution. An exemplary aqueous solution is 0.9% NaClsaline solution available from Baxter International (Deerfield, Ill.)and others.

In one embodiment, the aqueous solution further includes one or moreorganic or inorganic carboxylic acid-containing compounds (hereinaftercarboxylic acids or carboxylic acid compounds) which may or may notcontain a hydroxyl group on the alpha carbon, optionally titrated to aneutral pH using a suitable base (e.g., neutralized to a pH of about 6.5to about 7.5 using an alkali metal base such as sodium hydroxide orpotassium hydroxide), which can alter water demand, flowability, and/orviscosity of the bone graft substitute cement composition upon mixing.Exemplary carboxylic acids include glycolic acid and lactic acid.Preferred carboxylic acids have a single carboxylic acid group, from 1to about 10 total carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10carbon atoms including the carbonyl carbon), and 0-5 hydroxyl groups(e.g., 0, 1, 2, 3, 4, or 5) attached to the carbon chain. In oneembodiment, the mixing solution is a 0.6M solution of glycolic acidneutralized to a pH of 7.0 using NaOH. Reference to the carboxylic acidcompound herein encompasses both the free acid and salt forms.

It has been discovered, as set forth in Example 3, that the presence ofthe carboxylic acid component in the aqueous solution prior to gammaradiation sterilization can lead to inconsistent bone graft substitutecement properties, such as “drift” in cement setting time, due todegradation of the acid resulting from the radiation exposure. Thus, inone preferred embodiment, the carboxylic acid compound discussed abovein connection with the aqueous mixing solution is packaged as acrystalline powder (e.g., in free acid or salt form) with the remainingparticulate components of the kit, either in admixture with one or moreother powder components or in a separate container, rather than insolution. Using the acid component in powder form avoids degradation ofthe acid upon sterilization of the composition with gamma radiation.Alternatively, the carboxylic acid component is added to the aqueoussolution after the solution is sterilized by radiation so that thecarboxylic acid is not exposed to sterilizing radiation while insolution.

In one embodiment, the carboxylic acid for use in the invention isneutralized to a pH of about 6.5 to about 7.5 in solution using, forexample, an alkali metal base as noted above, and then isolated as acrystalline powder by evaporation of the solvent (e.g., water). Thecrystalline powder is typically isolated in a salt form, such as analkali metal salt form (e.g., lithium, sodium, or potassium salts).Exemplary dry crystalline powders of a carboxylic acid, in salt form,for use in the invention include sodium glycolate, potassium glycolate,sodium lactate, and potassium lactate. The powdered carboxylic acid saltcan be added to any of the other powder ingredients that together formthe particulate portion of the bone graft substitute cement, such as theCSH component or either of the calcium phosphate components. However, incertain embodiments, the powdered carboxylic acid is stored in aseparate container so that it can be reconstituted with the aqueoussolution prior to mixing the solution with the remaining particulatecomponents of the composition.

The bone graft substitute cement of the invention can further includeother additives known in the art. The additives can be added as a solidor liquid to either the particulate composition of the invention or theaqueous mixing solution. One example of an additive for the calciumsulfate composition is a plasticizer designed to alter the consistencyand setting time of the composition. Such a plasticizing ingredient canretard the setting of calcium sulfate hemihydrate pastes, therebyincreasing the time it takes for the composition to set following mixingwith an aqueous solution. Exemplary plasticizers include glycerol andother polyols, vinyl alcohol, stearic acid, hyaluronic acid, cellulosederivatives and mixtures thereof. Alkyl celluloses are particularlypreferred as the plasticizer ingredient. Exemplary alkyl cellulosesinclude methylhydroxypropylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetatebutyrate, and mixtures or salts thereof.

Exemplary additives also include biologically active agents. As usedherein, the term “biologically active agent” is directed to any agent,drug, compound, composition of matter or mixture that provides somepharmacologic affect that can be demonstrated in vivo or in vitro.Examples of biologically active agents include, but are not limited to,peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, polynucleotides, nucleic acids, cells,viruses, liposomes, microparticles, and micelles. It includes agentsthat produce a localized or systemic effect in a patient.

Particularly preferred classes of biologically active agents includeosteoinductive or osteoconductive materials, antibiotics,chemotherapeutic agents, pesticides (e.g., antifungal agents andantiparasitic agents), antivirals, anti-inflammatory agents, andanalgesics. Exemplary antibiotics include ciprofloxacin, tetracycline,oxytetracycline, chlorotetracycline, cephalosporins, aminoglycocides(e.g., tobramycin, kanamycin, neomycin, erithromycin, vancomycin,gentamycin, and streptomycin), bacitracin, rifampicin,N-dimethylrifampicin, chloromycetin, and derivatives thereof. Exemplarychemotherapeutic agents include cis-platinum, 5-fluorouracil (5-FU),taxol and/or taxotere, ifosfamide, methotrexate, and doxorubicinhydrochloride. Exemplary analgesics include lidocaine hydrochloride,bipivacaine and non-steroidal anti-inflammatory drugs such as ketorolactromethamine. Exemplary antivirals include gangcyclovir, zidovudine,amantidine, vidarabine, ribaravin, trifluridine, acyclovir,dideoxyuridine, antibodies to viral components or gene products,cytokines, and interleukins. An exemplary antiparasitic agent ispentamidine. Exemplary anti-inflammatory agents include α-1-anti-trypsinand α-1-antichymotrypsin.

Useful antifungal agents include diflucan, ketaconizole, nystatin,griseofulvin, mycostatin, miconazole and its derivatives as described inU.S. Pat. No. 3,717,655, the entire teachings of which are incorporatedherein by reference; bisdiguanides such as chlorhexidine; and moreparticularly quaternary ammonium compounds such as domiphen bromide,domiphen chloride, domiphen fluoride, benzalkonium chloride, cetylpyridinium chloride, dequalinium chloride, the cis isomer ofi-(3-chlorallyl)-3,5,7-triaza-1-azoniaadamantane chloride (availablecommercially from the Dow Chemical Company under the trademark Dowicil200) and its analogues as described in U.S. Pat. No. 3,228,828, theentire teachings of which are incorporated herein by reference, cetyltrimethyl ammonium bromide as well as benzethonium chloride andmethylbenzethonium chloride such as described in U.S. Pat. Nos.2,170,111; 2,115,250; and 2,229,024, the entire teachings of which areincorporated herein by reference; the carbanilides and salicylanilidessuch 3,4,4′-trichlorocarbanilide, and 3,4,5-tribromosalicylanilide; thehydroxydiphenyls such as dichlorophene, tetrachlorophene,hexachlorophene, and 2,4,4′-trichloro-2′-hydroxydiphenylether; andorganometallic and halogen antiseptics such as sinc pyrithione, silversulfadiazone, silver uracil, iodine, and the iodophores derived fromnon-ionic surface active agents such as described in U.S. Pat. Nos.2,710,277 and 2,977,315, the entire teachings of which are incorporatedherein by reference, and from polyvinylpyrrolidone such as described inU.S. Pat. Nos. 2,706,701, 2,826,532 and 2,900,305, the entire teachingsof which are incorporated herein by reference.

As used herein, the term “growth factors” encompasses any cellularproduct that modulates the growth or differentiation of other cells,particularly connective tissue progenitor cells. The growth factors thatmay be used in accordance with the present invention include, but arenot limited to, fibroblast growth factors (e.g., FGF-1, FGF-2, FGF-4);platelet-derived growth factor (PDGF) including PDGF-AB, PDGF-BB andPDGF-AA; bone morphogenic proteins (BMPs) such as any of BMP-1 toBMP-18; osteogenic proteins (e.g., OP-1, OP-2, or OP-3); transforminggrowth factor-α, transforming growth factor-β (e.g., β1, β2, or β3); LIMmineralization proteins (LMPs); osteoid-inducing factor (OIF);angiogenin(s); endothelins; growth differentiation factors (GDF's);ADMP-1; endothelins; hepatocyte growth factor and keratinocyte growthfactor; osteogenin (bone morphogenetic protein-3); heparin-bindinggrowth factors (HBGFs) such as HBGF-1 and HBGF-2; the hedgehog family ofproteins including indian, sonic, and desert hedgehog; interleukins (IL)including IL-1 thru -6; colony-stimulating factors (CSF) includingCSF-1, G-CSF, and GM-CSF; epithelial growth factors (EGFs); andinsulin-like growth factors (e.g., IGF-I and -II); demineralized bonematrix (DBM); cytokines; osteopontin; and osteonectin, including anyisoforms of the above proteins. Particulate DBM is a preferredosteoinductive additive.

The biologically active agent may also be an antibody. Suitableantibodies, include by way of example, STRO-1, SH-2, SH-3, SH-4, SB-10,SB-20, and antibodies to alkaline phosphatase. Such antibodies aredescribed in Haynesworth et al., Bone (1992), 13:69-80; Bruder, S. etal., Trans Ortho Res Soc (1996), 21:574; Haynesworth, S. E., et al.,Bone (1992), 13:69-80; Stewart, K., et al, J Bone Miner Res (1996),11(Suppl.):S142; Flemming J E, et al., in “Embryonic Human Skin.Developmental Dynamics,” 212:119-132, (1998); and Bruder S P, et al.,Bone (1997), 21(3): 225-235, the entire teachings of which areincorporated herein by reference.

Other examples of biologically active agents include bone marrowaspirate, platelet concentrate, blood, allograft bone, cancellous bonechips, synthetically derived or naturally derived chips of minerals suchas calcium phosphate or calcium carbonate, mesenchymal stem cells, andchunks, shards, and/or pellets of calcium sulfate.

A bone graft substitute cement according to the invention can be formedby mixing the particulate composition with the aqueous solution usingmanual or mechanical mixing techniques and apparatus known in the art.It is preferred to mix the components of the cement at atmosphericpressure or below (e.g., under vacuum) and at a temperature that willnot result in freezing of the aqueous component of the mixture orsignificant evaporation. Following mixing, the homogenous compositiontypically has a paste-like consistency, although the viscosity andflowability of the mixture can vary depending on the additives therein.The bone graft substitute cement material can be transferred to adelivery device, such as a syringe, and injected into a target site, forexample, to fill in cracks or voids of a bone defect. In someembodiments, the material can be injected through an 11 to 16-gaugeneedle up to, for example, 10 cm long.

The bone graft substitute cements of the invention will generally set,as defined by the Vicat needle drop test set forth below, in about 3 toabout 25 minutes, more preferably about 10 to about 20 minutes. The bonegraft substitute cement material of the invention will typically reach ahardness comparable to or greater than bone within about 30 to about 60minutes. Setting of the material can occur in a variety of environments,including air, water, in vivo, and under any number of in vitroconditions.

The hardened bone graft substitute cement preferably exhibits certainmechanical strength properties, particularly as characterized bydiametral tensile strength and compressive strength. Preferredembodiments of the cement exhibit a diametral tensile strength of atleast about 4 MPa after curing for one hour in ambient air followingmixing of the particulate composition with an aqueous solution, morepreferably a diametral tensile strength of at least about 5 MPa, mostpreferably at least about 6 MPa. Further, preferred embodiments of thebone graft substitute cement exhibit a diametral tensile strength of atleast about 8 MPa after curing for 24 hours in ambient air followingmixing of the particulate composition with an aqueous solution, morepreferably a diametral tensile strength of at least about 9 MPa aftercuring for 24 hours, and most preferably at least about 10 MPa.

Preferred embodiments of the bone graft substitute cement also exhibit ahigh level of compressive strength, such as a compressive strength of atleast about 15 MPa after curing for one hour in ambient air followingmixing of the particulate composition with an aqueous solution, morepreferably a compressive strength of at least about 40 MPa. Further,preferred embodiments of the bone graft substitute cement will exhibit acompressive strength of at least about 50 MPa after curing for 24 hoursin ambient air following mixing of the particulate composition with anaqueous solution, more preferably a compressive strength of at leastabout 80 MPa.

The bone graft substitute cement of the invention will also exhibit adissolution rate that is significantly slower than a comparable bonegraft substitute cement made substantially entirely of calcium sulfate.In certain preferred embodiments, the cement of the invention exhibitsan average dissolution rate, expressed as an average percentage ofweight loss per day, that is at least about 25% lower than the averagedissolution rate of a cement formed using a particulate compositionconsisting of calcium sulfate, the average dissolution rate measured byimmersion of a 4.8 mm OD pellet having a length of 3.3 mm in distilledwater at 37° C. as described in greater detail below. More preferably,the bone graft substitute cement of the invention has an averagedissolution rate that is at least about 30% lower than a calcium sulfatecement, most preferably at least about 35% lower, and in someembodiments, as much as 40% lower or more. A preferred range ofdissolution, expressed as an average percentage of weight loss per daymeasured using the test procedure set forth below, is about 5% to about15%, more preferably about 7% to about 13%. Average dissolution ratesstated are determined by linear regression of % weight loss per dayusing data from days 0, 1, 2, 3, and 4 determined using the procedureset forth below.

The present invention also provides a bone graft substitute kitcomprising the particulate composition of the invention. Typically, thekit comprises one or more containers enclosing the particulatecomposition as described above and a separate container enclosing asterile aqueous solution. The kit will typically contain a writteninstruction set describing a method of using the kit. In addition, thebone-graft substitute kit of the invention will preferably comprise anapparatus for mixing the particulate composition with the aqueoussolution in order to form the bone graft cement, such as a vacuum mixingapparatus. Additionally, the kit will typically include a device fordelivering the bone graft cement to the site of the bone defect, such asan injection device (e.g., a needle and syringe). The particularcomposition and the sterile aqueous solution will typically besterilized by irradiation prior to packaging in the kit.

As noted previously, in certain embodiments, the kit of the inventionwill separate the two calcium phosphate powder components into differentcontainers to avoid reaction during storage. There are a number ofpackaging configurations that can accomplish this goal. For example, inone embodiment, the kit includes one container for CSH powder, onecontainer for β-TCP powder, and one container for MCPM powder. Inanother embodiment, the kit includes two containers for the particulatecomposition, one including β-TCP powder and a portion of the CSHcomponent and a second containing MCPM powder and a portion of the CSHcomponent. In yet another embodiment, the MCPM powder is packaged in aseparate container by itself, and the β-TCP powder and the CSH powderare packaged together. In a still further embodiment, the β-TCP powderis packaged in a separate container by itself, and the MCPM powder andthe CSH powder are packaged together. In any of the above embodiments,any of the powder containers can further include the crystalline powderof the carboxylic acid salt component and/or the β-TCP granules, orthose components could be packaged separately in their own containers.When present, the accelerator adapted to accelerate conversion of CSH toCSD is typically in admixture with the CSH powder. In one preferredembodiment, the kit comprises one container enclosing the MCPM powder,and a second container enclosing the remaining particulate ingredientsin admixture, such as one or more of the CSH powder, the CSHaccelerator, the β-TCP powder, the β-TCP granules, and the carboxylicacid crystalline powder.

In one preferred embodiment, the powdered form of the carboxylic acid ispackaged separately so that it can be reconstituted in the aqueoussolution, if desired, prior to mixing the solution with the remainingparticulate components. However, as noted previously, the aqueoussolution of the kit may also contain the carboxylic acid component insolution form if the carboxylic acid is added after radiationsterilization of the aqueous component of the kit.

It can be important to utilize all of the aqueous solution packaged inthe kit in order to ensure that consistent setting times are achieved.In one embodiment, the aqueous solution is packaged in a highlyhydrophobic container, such as a glass syringe or other glass container,that is less prone to retention of residual solution in amounts thatwill cause changes in the performance characteristics of the bone graftsubstitute cement.

The present invention also provides a method for treating a bone defect.The method of the invention involves applying a bone graft substitutecement as described above to the site of the bone defect. The bone graftsubstitute cement can be applied in flowable form following mixing ofthe particulate composition with the aqueous solution, such as throughan injection device, prior to setting of the composition. Alternatively,the bone graft substitute cement can be used in a precast hardened form,wherein the cement is provided in predetermined shapes such as pellets,granules, wedges, blocks, or disks, or used in the form ofrandomly-shaped shards created by mechanically breaking a cement massinto smaller pieces. In a further embodiment, the clinician can form thebone graft cement mixture and manually mold the mixture into a desiredshape, such as the shape needed to fill a particular bone defect, priorto application.

In another embodiment, the bone graft substitute cement of the inventioncan be incorporated into an orthopedic implant, such as any of thevarious devices adapted for joint replacement. The bone graft substitutecement is typically incorporated into such devices as an outer coatingor as a filling material within the pores of a porous outer component ofthe device. In this embodiment, the bone graft substitute cementfacilitates bone ingrowth in the area surrounding the implanted device.Exemplary orthopedic implants include knee replacement devices (e.g.,constrained or non-constrained knee implant devices, hinged kneedevices, metallic plateau knee devices, and patellar devices), hipreplacement devices (e.g., acetabular components and femoralcomponents), elbow replacement devices (e.g., constrained,semi-constrained, and non-constrained devices), upper femoral devices,upper humeral devices, wrist replacement devices (e.g., semi-constrained2- and 3-part articulation devices), shoulder devices, passive tendondevices, spinal devices (e.g., thoracolumbar spinal fixation devices,cervical spinal fixation devices, and spinal fusion cages), finger/toedevices, and diaphysis devices.

The present invention will be further illustrated by the followingnon-limiting example.

EXPERIMENTAL

Example 1 illustrates in vivo use of a bone graft substitute cement ofthe invention, and particularly describes the reduced resorption rate(as compared to a calcium sulfate composition), good mechanicalproperties, and acceptable setting times exhibited by the inventivecomposition. Example 2 illustrates the ability of an embodiment of theinventive composition increase the amount, strength, and stiffness ofrestored bone as compared to use of conventional CaSO₄ pellets. Example3 demonstrates the degradation effect of gamma radiation on glycolicacid in solution, and the effect of such degradation on setting times ofthe bone graft substitute cement. Example 4 demonstrates that placementof a glycolic acid salt form in the particulate composition reduces theeffect of radiation on the performance of the bone graft substitutecement without sacrificing other advantageous properties, such ascertain handling and mechanical strength properties.

Setting Time Measurement

Setting times can be measured using a Vicat needle that is 1 mm indiameter, 5 cm long, and which possesses a total weight of 300 g, allper ASTM C-472, which is incorporated by reference herein in itsentirety. The sample being tested should be mixed in a manner that ahomogeneous, flowable paste is created. The sample size for the Vicatneedle drop test is about 3 cc to about 5 cc of material tapped down toa cake in an approximately 20 mL polyethylene cup; the sample shall behandled such that no agitation is inflicted upon the material 1 minuteafter the aqueous solution contacts the particulate composition otherthan the dropping and removal of the Vicat needle. The cup should be ofsuch dimensions that the cake is a short, flat cylinder measuring about¼″ to about ⅜″ in height.

Set time according to the Vicat needle drop test is defined as theamount of time elapsed between the time the aqueous solution contactsthe particulate composition and the time the Vicat needle will not passthrough 50% of the height of a cement sample upon being dropped from theupper surface of the sample. The needle is allowed to fall under its ownweight, under gravity alone, through a line perpendicular to the top andbottom, flat faces of the cylinder-shaped sample cake. The needle isdropped every 30 seconds after the first drop. The needle shall not bedropped more than 6 times during the duration of the test. If after the6^(th) drop the needle continues to pass through more that 50% of theheight of the sample, the test must be repeated with fresh material; anew, clean cup; and a clean Vicat needle free of debris, especially thatwhich is left behind from previous tests. Cups, mixing equipment, andmaterial transfer equipment should not be reused. All materials andequipment used during testing should be between 21-27° C. and exposed toan environment with a relative humidity between 20-50%.

Compression Strength Measurement

Compression strength of the material is determined through the followingtest methodology. Specimens are cast to size per ASTM F451 (6 mm outerdiameter×12 mm in length), which is incorporated by reference in itsentirety, utilizing a stainless steel split mold with a capacity ofeight specimens.

The split mold is placed on a glass plate with the cylindrical voids,specimen slots, standing upright. The material is mixed and then loadedinto a device for delivery of the material into the slots such that aback filling method can be utilized; a syringe with a jamshidi-typeneedle is commonly used. Each specimen slot is filled from bottom to topin a back filling manner. It is customary to excessively fill the moldsuch that excess material extrudes out above the dimensions of the splitmolds, this assures displacement of any air entrapped within thespecimen slots. It may be necessary to hold mold down on to glass plateduring casting to prevent material from extruding out of the bottom ofthe specimen slots, between the glass plate and mold.

Upon filling each specimen slot another glass plate is pushed by handonto the excess material located on the top of the mold, producing athin sheet of flashing across the tops of the specimens and split molditself. This glass plate is of a size which does not produce anexcessive compressive force or a pressurized environment in which thematerial cures. All specimens are cast and flashing is created within 2minutes of the aqueous solution coming into contact with the particulatecomponent.

The specimens are demolded 30 minutes after the aqueous solution hascome into contact with the particulate component. First the flashing isremoved from both sides of the split mold containing the faces of thespecimens; regardless of holding mold against the lower glass plate uponcasting, a thin film of flashing is created on the lower surface of themold. Commonly, a razor blade is used to scrape off the flashing and indoing so create smooth faces on the specimens. The split mold isseparated and the specimens are removed. All specimens should be removedwithin 32 minutes of the aqueous solution coming into contact of theparticulate component. Upon removal of the specimens, they should beallowed to continue curing in air at room condition (21-27° C.; 20-50%relative humidity) until time of testing.

Testing of the material is performed at a predetermined time after theaqueous solution has come into contact with the particulate component.Commonly, testing is performed at 1 hr and 24 hrs. Testing is performedon a compression test fixture per ASTM D695, which is incorporated byreference herein in its entirety. The compression test fixture is placedon a mechanical test frame capable of displacement control andmonitoring of displacement and force through data acquisition running at50 Hz or faster.

The specimens are tested individually on the compression test frame. Thespecimens are placed between the platens in a manner such that thecylinder faces are positioned against the platens. The compression testframe containing the specimen is loaded in compression at a rate of0.333 mm/sec until failure. Force and displacement are monitoredthroughout the test, and maximum force at failure is noted. Properfailure will result in a fracture across the height of the specimen. Themaximum compression force at failure is noted. Failure is defined as asudden drop in load, deviation of the loading curve from the initialslope created by the loading of the specimen, and/or the force notedupon visual failure of the specimen.

The compression strength in MPa is then calculated as followed:(Pmax)/(π*R²); where Pmax is the load at failure in Newtons, π isapproximately 3.14, and R is the radius of the specimen in mm (3).

It is crucial when performing compression strength specimen preparationthat all equipment used is clean of all debris, especially that of thecured material of interest.

Diametral Tensile Strength Measurement

The diametral tensile strength is determined through the following testmethodology. A 1″ cube of 10 lb/ft³ closed-cell polyurethane foam(available as Last-A-Foam® from General Plastics Manufacturing Company,Tacoma, Wash.) with an approximately ⅝ in. (15.8 mm) outer diametercylindrical void and notches for side removal is used as the specimenmold. The approximately ⅝ in. outer diameter cylindrical void is createdby drilling perpendicularly through opposite faces of the cube in onedepression of a drill press utilizing a ⅝ in. drill bit. The void runsthe entire length of the cube and is centered such that both opposite,drilled faces share the same center as the circular voids created inthem from the drilling. Two opposite sides from the remaining four fullsides are designated to become the open sides of the final specimen;these sides will be removed via the notches. These sides are notched,two notches per side, in a manner such that they can be removedimmediately prior to testing and not affect the sample integrity. Thenotches shall run the entire length of the cube and be separated in amanner that upon removal >50% of the height of the specimen is exposed.Commonly the notches are created using an upright band saw. FIGS. 2 a-2c illustrate an exemplary diametral tensile test mold 20. FIG. 2 aprovides a top and bottom view of the mold 20. FIG. 2 b provides a sideview of the mold 20. FIG. 2 c provides a front and rear view of the mold20 and shows a 16 mm outer diameter cylindrical void 30 therein.

The material to be tested is mixed to a homogeneous paste and loadedinto a device suitable for injection of the paste into the 16 mm outerdiameter cylindrical void. Commonly a 30 cc syringe with a 1 cm openingis used for this. The mold is held by hand using the thumb and middlefinger positioned on the opposite, notched sides. The index finger ofthe hand used to hold the mold is positioned over one of the circularopenings. The material is then injected into the void from the oppositeside of the void from the index finger; the entire face of the syringeexhibiting the 1 cm opening is lightly pushed up against the circularopening of the mold. Upon injection of the material into the mold,pressure will be felt on the index finger covering the back opening fromthe ejected material. The index finger is slowly removed while fillingcontinues, allowing the paste to flow out of the rear of the mold in anextrusion with the same 16 mm outer diameter as the void. The syringe isslowly backed out from the front opening while back filling of paste isperformed through further ejection from the syringe until the entirevoid is filled and excess material is located outside the dimensions ofthe original cube of foam. The front and rear sides of the specimen arewiped smooth, flush with the front and rear sides of the mold using aspatula. All specimens to be tested should be made within 2 minutes fromthe start of mixing, defined by the aqueous solution coming into contactwith the particulate composition.

The specimens are allowed to cure horizontally in air in the mold withthe front and rear sides of the mold exposed to air at room conditions(21-27° C.; 20-50% relative humidity) for a predetermined amount oftime, normally 1 hr or 24 hrs. This predetermined amount of time beginsat the time at which the aqueous solution comes into contact with theparticulate composition at the beginning of the mixing process.

Testing is performed on a mechanical test frame capable of displacementcontrol and of monitoring displacement and force through dataacquisition running at 20 Hz or faster. The sides of the specimen moldare removed immediately prior to testing; only the areas between thenotches are removed.

Removal of the sides is normally performed with a knife. The top andbottom of the mold are held between two fingers with slight pressure toprevent specimen surface-to-mold interface damage. The knife blade isplaced into one of the notches and then twisted to break the areabetween the notches free; this is repeated for the other side in thesame manner. The tops and bottom of the molds are left in place to holdthe specimen and prevent shear stresses on the surface. The specimen isplaced between two flat, parallel platens; one of which is free toswivel to allow alignment with the loading train. The swiveling platenassures an equally distributed load across the specimen contact points.The specimen is loaded transversely at a rate of 5 mm/minute untilfailure. Proper failure will result in a vertical fracture completelythrough the length of the specimen. The maximum force at failure isnoted.

A loading curve of force versus displacement is created to determine themaximum force at failure, in which displacement and force are positivevalues. The first part of the loading curve shows the loading of thefoam followed by its compression. The compression of the foam portionwill be evident by continued displacement with no substantial increasein force; this can also be seen visually during the test. After the foamis completely compressed, the force will begin to rise again, creatingan increasing slope on the loading curve followed by a constant slope asthe load is transferred to the specimen. The increasing slope iscommonly known as a “toe in”. Failure is defined as a sudden drop inload, a decrease in the slope of the loading curve after the constantslope from specimen loading has been established, and/or the force notedupon visual failure of the specimen while the test is running.

The diametral tensile strength in MPa is then calculated as followed:(2*Pmax)/(π*L*H); where Pmax is the load at failure in Newtons, π isapproximately equal to 3.14, L is the length of the specimen in mm(25.4), and H is the height of the specimen in mm (16). Specimens aredisqualified for diametral tensile strengths if any one or more of thefollowing occur: fracture is not vertical, facture does not completelyrun the length of the specimen, length of the specimen fails, or voidsin the material are seen on the fractured walls of the specimen.

It is crucial when performing diametral tensile strength specimenpreparation that all equipment used is clean of all debris, especiallythat of the cured material of interest.

Dissolution Rate Measurement

Dissolution rate of the material is determined through the followingmethodology. Specimens are cast in silicone molds to a size of 4.8 mmouter diameter and 3.3 mm tall cylinders. A 3.3 mm thick sheet ofsilicone containing cylindrical voids is used as a mold. Cylindricalvoids are 4.8 mm in outer diameter and 3.3 mm tall, and orientated suchthat the circular faces of the void are parallel and in the same planeas the surfaces of the silicone sheet.

A thin sheet of polyethylene is laid on a table. A polyethylene mesh isplaced on top of the polyethylene sheet; sheet and mesh are of samedimensions (excluding thickness) and positioned such that the mesh masksthe sheet from the top. Next a silicone mold of smaller dimensions isplaced on top of the mesh (excluding thickness). No part of the moldhangs off the edge of the mesh or sheet.

The material to be tested is then mixed together to form a homogeneouspaste. The paste is then wiped across the top of the mold using aspatula in a manner that the voids are packed with the material. Themesh will allow air to be displaced out of the void as the mold isfilled. Several wipes are performed to assure that material hascompletely penetrated to bottom of the mold and extruded out through themesh and onto the lower polyethylene sheet. A final wipe with thespatula across the top of the mold is performed to remove the majorityof excess material and produce smooth top faces for the specimens.

Another polyethylene sheet of the same dimensions of the as the first isthen placed across the top of the mold, such that it completely coversthe top of the mold. This sheet is then gently pressed against the moldusing a finger in a gentle rubbing motion. An intimate contact betweenthe top polyethylene sheet and the specimens is created.

The entire system, sheet, mesh, mold, and sheet, is then picked up as awhole and flipped over in a manner such that the original top is nowfacing down. The system is held by hand and slapped repeatedly ontotable in a manner such that any air entrapped in the molds will bedisplaced out by the material; slapping of the system should not beexcessive in force or repetitions. Upon removal of the majority of theair the system is returned to table in the upside down orientation,sheet and mesh side up. The top polyethylene sheet, originally thebottom, and mesh are removed and the spatula is again used to wipematerial into voids in the tops (previously bottoms) of the specimenscreated from air removal. A final wipe with the spatula across the topof the mold is performed to remove the majority of excess material. Thesheet (no mesh) is returned to the top of the mold. The sheet is thenpressed against the mold using a finger in a gentle rubbing motion. Anintimate contact between the top and bottom polyethylene sheet and thespecimens has now been created.

The specimens are left in the mold to cure for a minimum of 8 hrs afterthe second polyethylene sheet has been placed in direct contact with thespecimens and mold (no mesh). After at least 8 hrs have passed, thespecimens are demolded by hand. Any flash remaining attached to pelletfaces are removed by rolling specimen between fingers. All defectivespecimens are disqualified from the test and discarded. A defectivespecimen is defined as a specimen not exhibiting a cylindrical shape,which could be caused by entrapped air, defects created upon demolding,and/or physical damage to the specimen itself.

All specimens which are not defective are spread across a stainlesssteel pan in a monolayer. The pan and specimens are then dried in anoven at 40° C. for a minimum of 4 hrs, and then removed from oven andallowed to cool for 30 minutes in room conditions (21-27° C.; 20-50%relative humidity).

From the specimens created, five (5) specimens are arbitrarily chosen tobe used for dissolution testing. Each specimen chosen is paired with aclean cylindrical fritted glass extraction thimble of the followingdimensions: 90.25 mm overall height, 4 mm fritted glass base (40-60micron pores) located 80 mm from top of thimble, 25 mm outer diameter,and 22 mm inner diameter. The mass of each extraction thimble ismeasured (0.01 mg) and noted. The mass of each specimen in measured(0.01 mg) and noted. A polyethylene bottle (300 mL) is designated toeach pair (specimen and thimble). The bottle has dimensions that allowthimble and specimen to easily be placed in and removed from bottle andupon filling with 275 mL of water will create a column of water that istaller than the thimble. The bottle is filled with 275 mL of distilledwater at room temperature (21-27° C.). The specimen is placed into itscorresponding thimble and the thimble is lowered into the bottle; careis taken to keep any part of the material from escaping from thethimble. The bottle is capped and placed into a water bath at 37° C.with no agitation and the time is noted.

24 hrs after the specimen has been in the water, the thimble containingthe specimen is retrieved. The water is allowed to drain out of thethimble through the fritted glass base. The thimble containing thespecimen is then dried for 4 hrs in a 40° C. oven or until completelydried (determined gravimetrically). The thimble containing the specimenis then allowed to cool down for 30 minutes at room conditions (21-27°C.; 20-50% relative humidity).

The thimble-containing the pellet is then weighed to an accuracy of 0.01mg. Subtracting the known empty thimble mass from the mass of thecombination will result in the mass of the specimen alone. Subtractingthis mass from the initial specimen mass will produce the mass lost todissolution. This mass lost can be divided by the specimen initial massand the product of that multiplied by 100 will result in the % mass lostfrom dissolution.

At this point the thimble containing the pellet is returned to thebottle containing fresh distilled water (275 mL) at room temperature(21-27° C.), and the bottle is capped and returned to the water bath.After 24 hrs the drying and weighing process is repeated. These actionsare repeated with fresh distilled water after every 24 hr soak until thetest is terminated or the material completely dissolves.

Example 1

Diametral tensile strength, dissolution properties, and in vivoevaluation of new bone ingrowth and residual material of bone graftcements of the invention were compared to a commercially availablecalcium sulfate material. The experimental group for all experiments wasan embodiment of the current invention including a cement consisting of74.906 weight percent calcium sulfate hemihydrate, 0.094 weight percentaccelerator (sucrose coated calcium sulfate dihydrate), 6.7 weightpercent monocalcium phosphate monohydrate, 8.3 weight percent betatricalcium phosphate powder, 10 weight percent beta tricalcium phosphategranules, and an aqueous solution of 0.6 molar glycolic acid neutralizedto a pH of 7.00 with 10 normal sodium hydroxide solution (hereinafter“SR”). MIIG® X3 Bone Graft Substitute (hereinafter “X3”) (WrightMedical, Arlington, Tenn.) calcium sulfate was used as a control for allexperiments. The SR material was formulated to set in 14-19 minutes,whereas the X3 material was formulated to set in 7-10 minutes.

An intermediate resorbing calcium sulfate, calcium phosphate compositecement was also evaluated in this study. Dissolution properties,compression strength, and in vivo evaluation of new bone ingrowth andresidual material were evaluated for this material. This material isalso an embodiment of the present invention and comprised 84.999 weightpercent calcium sulfate hemihydrate, 6.7 weight percent monocalciumphosphate monohydrate, 8.3 weight percent beta tricalcium phosphatepowder, 0.0013 weight percent accelerator (sucrose coated calciumsulfate dihydrate), and an aqueous component of water. This intermediatematerial was formulated to set in 11-16 minutes.

Compression strength was measured on vacuum mixed specimens cast in themanner set forth above. Specimens (n=6) were cured for 1 hr in ambientair. Specimens (n=3) were cured for 24 hrs in ambient air. The specimenswere loaded lengthwise using a MTS 858 Bionix test system at a constantrate of 0.333 mm/sec. The compression strength in MPa was calculatedusing the formula (Pmax)/(π*R²).

Diametral tensile strength (DTS) was measured on vacuum mixed specimenscast in the manner set forth above. The sides of the foam blocks wereremoved prior to testing. Specimens (n=4) were cured for 1 and 24 hrs inambient air at room temperature. The specimens were transversely loadedto failure in compression using a MTS 858 Bionix test system at aconstant rate of 5 mm/min. DTS was calculated from the formulaDTS=(2*Pmax)/(π*L*H).

Dissolution tests were performed on 4.8 mm OD X 3.3 mm cylindricalpellets (n=5). Specimens were placed in 275 mL of distilled water at 37°C. Solutions were changed daily. Specimens were dried and weighed dailyfor first 30 days and every 5 days thereafter until a residual mass of<5% was achieved. X-Ray diffraction (XRD) was used to identify theresidual material.

Results:

FIG. 3 shows the DTS results. One-way ANOVA were performed using JMPsoftware (SAS, Cary, N.C.). A significant difference was seen between 1and 24 hr cure times for SR cured in air (p<0.001) and no difference forthe X3 (p=0.508). It is apparent from the air cured data that the SRreaction is incomplete at 1 hr while the X3 setting reaction isessentially complete. This result was expected based on the differencesin setting time.

Average, maximum, and minimum compression strength values for theintermediate material were determined. The 1 hr cure time data producedan average strength of 19.4 MPa, a minimum of 16.2 MPa, and a maximum of21.4 MPa. The 24 hr cure data produced an average strength of 69.9 MPa,a minimum of 61.4 MPa, and a maximum of 77.3 MPa.

Dissolution results are shown in FIG. 4. Linear regression of days 0through 4 of the curves were used to estimate the dissolution rates. Theaverage SR rate was 10.7%/day, while the X3 rate was 17.8%/day. Theaverage rate for the intermediate material was 13.5%/day. Followingdissolution of 95% of the bone graft substitute cement material, XRD ofresidual SR material showed it to be beta tricalcium phosphate, a knownbioresorbable and osteoconductive material.

A 6-week in vivo pilot study was conducted under an Institutional AnimalCare and Use Committee (IACUC) approved protocol. In each of 3 dogs, twodefects measuring 9 mm×15 mm were created in each proximal humerus. Eachsite was filled with either an injected bolus of SR (1-1.5 cc), 4.8 mmOD X 3.3 mm pellets of SR, 4.8 mm OD X 3.3 mm pellets of X3, or aninjected bolus of the intermediate resorbing calcium sulfate, calciumphosphate composite cement. Implants were sterilized with gammaradiation. Each dog received one implant of each material. Healing ofthe defects and resorption of the pellets and boluses were assessed fromradiographs obtained after 0, 2, and 4 weeks and contact radiographsafter 6 weeks. New bone formation and residual implanted material in thedefects were evaluated using light microscopy of undecalcified, plasticembedded histological sections stained with basic fuchsin and toluidineblue. Area fraction of new bone and residual material in the defectswere determined using histomorphometry.

In the in vivo study, the radiographic and histologic data indicatedthat both types of pellets and boluses were replaced with newly formedosteoid, woven, and lamellar bone that had formed in concentric lamellaeat the previous implant sites. At 6 weeks, area fraction of new boneformation was 35.9±6.1% for defects implanted with SR pellets and26.7±10.0% for defects implanted with X3 pellets. At 6 weeks, themajority of the implanted pellet materials had resorbed, but there wasslightly more residual implant material in SR pellet defects compared tothe X3 pellet defects. For the SR bolus implants new bone formation was15.6±5.6% with 29.9±11.9% residual implant material. For the bolus ofintermediate resorbing calcium sulfate, calcium phosphate compositecement, new bone formation was 23.4±7.1% with 19.3±8.0% residual implantmaterial. Smaller fractions of new bone formation can be expected forbolus materials at early time due to larger percentages of residualmaterial and smaller surface area to implant volume ratios when comparedto that of pellets.

The composite cement of the invention demonstrated consistent settingand strength characteristics similar to those of the control. The goalof slowing down the dissolution rate was achieved, and the early in vivobone growth was equivalent or superior to the pure calcium sulfatecontrol.

Example 2

Materials and Method:

Under an IACUC-approved protocol, 10 skeletally mature, male dogs (25-32kgs) had a critical-size, axial medullary defect (13 mm dia×50 mm)created bilaterally in the proximal humerus and were studied for 13(n=5) and 26 (n=5) weeks. The defect in one humerus was injected with 6cc of the test material (SR cement according to Example 1). An identicaldefect in the contralateral humerus received an equal volume of CaSO₄pellets (OSEOSET® pellets, Wright Medical). Radiographs were obtained at0, 2, 6, 13 and 26 weeks. Transverse, undecalcified stained sections ofthe bones were prepared. The area fractions of new bone and implantedresidual materials in the defects were quantified using standardpoint-counting techniques. The sections were also examined usinghigh-resolution contact radiographs. The yield strength and modulus ofan 8 mm dia.×20 mm test cylinder cored from the midlevel of each defectwas determined in unconfined, uniaxial compression tests at a crossheadspeed of 0.5 mm/min. The histomorphometric and biomechanical data wereanalyzed using the Friedman and Mann-Whitney tests. Data are presentedas the mean and standard deviation.

Results:

The clinical and postmortem radiographs revealed markedly differentrates of resorption of the bone graft substitutes and replacement withbone in the defects. Resorption of the CaSO₄ pellets was apparentbeginning at 2 weeks and substantially complete by 6 weeks. There wasslower resorption of the SR cement, also beginning at 2 weeks, but somecement persisted at 26 weeks.

In all of the stained histological sections, there was restoration ofthe defects by bone and marrow with only focal areas of fibrous tissueand relatively low volumes of residual implanted material. The areafraction of new mineralized bone at 13 weeks was 2-fold greater indefects treated with SR cement (39.4±4.7%) compared to defects treatedwith conventional CaSO₄ pellets (17.3±4.3%) (p=0.025). At 26 weeks, thebone had remodeled to a more normal architecture, but there was stillmore bone in defects treated with cement (1 8.0±3.4%) compared topellets (11.2±2.6%) (p=0.025).

Residual matrix and β-TCP granules were incorporated into bonetrabeculae. Surfaces of the materials not covered by bone appeared to beundergoing remodeling by osteoclast-like cells, some of which containedminute particles. The area fraction of residual matrix was greater inthe cement-treated defects at 13 weeks (2.9±2.8%) and at 26 weeks(0.6±0.8%) compared to pellet-treated defects (0.0% at 13 and 26 weeks)(p=0.025 and 0.083, respectively). Residual matrix decreased with timein the cement-treated defects (p=0.047). The area fraction of residualβ-TCP granules also decreased from 13 weeks (3.6±1.0%) to 26 weeks(0.8±1.4%) (p=0.016). The maximum dimension of the β-TCP granulesdecreased from 348±13 gm at 13 weeks to 296±29 μm at 26 weeks (p=0.008).

Cored bone samples from defects treated with the cement wereconsiderably stronger and stiffer than those treated with CaSO₄ pelletsat both 13 and 26 weeks (Table 1 below). For comparison, similar coredtrabecular bone specimens from 8 normal proximal humeri had a yieldstrength of 1.4±0.66 MPa and a modulus of 117±72 MPa. TABLE 1 Time (wks)SR Cement CaSO₄ Pellets Yield Strength (MPa) 13  5.3 (2.6)* .90 (.44)Yield Strength (MPa) 26  2.2 (.41)** .47 (.46) Modulus (MPa) 13 283(217) 40.8 (35.6) Modulus (MPa) 26 150 (73)* 15.8 (23.6)*p = 0.025,**p = .046, different from pelletsConclusion:

Several Ca-based materials with different resorption rates weresuccessfully combined to produce a cement with a tailored, slowerresorption profile. In this cement, the majority of the calcium sulfateand dicalcium phosphate dihydrate matrix resorbs early, promoting boneformation deep into the bolus of cement, while the distributed β-TCPgranules provide a scaffold, incorporate into new bone, and are thenmore slowly resorbed. The engineered cement increased the amount,strength and stiffness of restored bone when compared to conventionalCaSO₄ pellets after 13 and 26 weeks. This cement holds promise forclinical applications where a strong, injectable and highlybiocompatible bone graft substitute would be advantageous.

Example 3

Materials and Method:

250 mL of mixing solution, 0.6M glycolic acid neutralized with sodiumhydroxide, was created and the pH was noted with a calibrated pH meter.The solution was made using crystalline glycolic acid (Alfa Aesar Part #A12511; Ward Hill, Mass.), 10N sodium hydroxide solution (EMD ChemicalsPart # SX0607N-6; Darmstadt, Germany), and USP water for irrigation(Baxter Healthcare Corporation Part # 2F7112; Deerfield, Ill.).

The solution was then divided into two 125 mL aliquots and thenindividually rebottled. One of the bottles was sent out for bulk gammaradiation sterilization, 25-32 kGy dose, and the other was retained asan unsterilized control. Upon return of the sterilized solution the pHof both the sterilized and the non-sterilized solutions were checkedwith a calibrated pH meter and noted.

A single lot of SR powder of the type utilized in Example 1 was used inthis study to avoid lot-to-lot variability in set time and injectionforces.

Three vials were filled with 6.9 mL of the unsterilized solution andcoupled with three vials of unsterilized SR powder containing 30 g pervial. This group served as a control.

Another group was made to represent the option of aseptic filling of theindividual units of neutralized glycolic acid. This group consisted ofthree vials of 6.9 mL of glycolic acid filled out of the 125 mL of bulksterilized solution and three vials of SR powder filled to 30 g. Thepowder vials were sent out for gamma radiation sterilization. Thisrepresents sterilization of the bulk solution followed by asepticfilling and coupling in a kit containing the already sterilized unit ofpowder.

The third and final group represents a preferred manufacturingsituation: gamma radiation sterilization of the bulk solution followedby gamma radiation sterilization of the individual units. Three vials ofsolution were filled to 6.9 mL with the sterilized bulk solution.Another three vials were filled with 30 g of the SR powder. All six ofthese vials were sent out to sterilization. This represents filling thesolution from a bulk sterilized solution, packaging kits containingunsterilized powder with bulk sterilized solution, and then sending thekit out for a final sterilization.

Upon return of all the groups the following testing was performed. Allsolutions, including the remainder of the bulk solution were checked forpH with a calibrated pH meter and noted. The nine sets of units (threeunits of unsterilized solution and unsterilized powder, three units ofone time bulk sterilized solution and unit sterilized powder, and threeunits of two times sterilized solution (once in bulk followed by once asa unit) and one time unit sterilized powder) were mixed to form ahomogeneous paste under vacuum. Set times of approx. ¼ in. thick aliquotof paste in a 25 mL plastic cup were determined through the use of a 300g Vicat needle. Injection force from a 3 cc syringe attached to a 6 cm11 gauge non-tapered, ported jamshidi type needle was determined at 3and 5 minutes after the powder and solutions had come into contact withone another. Injection forces are reported as forces seen at 15 mm ofplunger displacement being displaced at 4.4 mm/sec. Injection testingwas performed using a materials test frame in displacement control, anddata acquisition was taken at 50 Hz of force and displacement.

Results:

pH drift was seen for all solutions. Results were consistent within agroup although the two time sterilized solution produced a pH differentfrom that of the control and one time sterilized group. Specifically,the two times sterilized solution produced an average pH of about 6.3,while the other solution groups exhibited a pH of about 5.5.

Forces of injection for all groups were the same. At the 3 minute timepoint the injection force was about 25 N, and for the 5 minute timepoint the injection force was about 40 N.

The set time for the unsterilized and one time sterilized group wereconsistently around 18.5 minutes, except for one unit of the one timesterilized group which was at 19.75 minutes. Set time measurements forthe two times solution sterilized group and one time powder sterilizedhad consistently shifted to about 22 minutes.

Conclusion:

The pH and set time shifts in the two times sterilized solution groupshows degradation of the neutralized glycolic acid solution throughgamma radiation sterilization. Although the effects were not pronouncedin the one-time sterilized solution, degradation must have occurred inthat group as radiation degradation is an additive process.

Example 4

Materials and Method:

First, the effect of gamma sterilization of crystalline glycolic acid(GA) on the material's acid-base titration curve using a stock solutionof 0.6M sodium hydroxide (NaOH) was examined. Then, physical propertycomparisons were made between radiation sterilized samples of a cementpowder with solid sodium glycolate (Na-GA) blended into the precursorpowder and a non-irradiated material. Diametral tensile strength,injection force, Vicat set time, and morphological (SEM) comparisons ofthe set cements from each configuration were made. Additionally, Vicatset time comparisons were made between unsterilized samples of eachproduct configuration.

Approximately 50 g of GA (GLYPURE® available from Dupont) was subjectedto gamma radiation sterilization (25-32 kGy dosage). Two ˜1M solutionsof GA were created at equal volumes, one with the gamma irradiated GAand the other with non-irradiated GA of the same manufacturing lot. Inorder to avoid loss of material during liquid transfers and fromevaporation, the solutions were made immediately prior to being used bydissolving 3.803 g of GA with 50.000 g of DI water in a 250 mL beaker.

A 500 mL 0.6M NaOH stock solution was created by diluting 30 mL of the10N NaOH with DI water in a 500 mL volumetric flask. This stock solutionwas used as the titrant for both GA solutions.

A 50 mL burette (0.1 mL increments) equipped with a stopcock was used todispense the NaOH stock solution in various increments directly into the250 mL beakers containing the ˜1M G solutions. During titration the GAsolutions were stirred using a polytetraflouroethylene coated magneticstir bar and plate. The volume of NaOH stock dispensed was monitored andrecorded through the titrations. The pH of the GA solution was alsomonitored and recorded with each increment of NaOH stock added. pHmeasurements were determined through use of a pH meter (VWR Scientific;Model 8000) and electrode (VWR Scientific, P/N 14002-780) calibratedbetween pH=4.00 and 7.00 using standard buffer solutions (VWRScientific, P/N 34170-130 and 34170-127, respectively). Titration wascarried out until minimal changes of pH in the alkaline range were seenwith consecutive additions of the stock solution. Titration curves (pHof GA solution vs. mL 0.6M NaOH) were plotted and comparisons were madeto detect effects of gamma irradiation on crystalline GA.

A 300 g batch of an SR material as described in Example 1 (Configuration1 with NA-GA in solution) was blended for 20 min in a 1 qt acrylicV-shell using a 60 Hz P-K Twin-Shell Yoke blender (Patterson-Kelley Co.;East Stroudsburg, Pa.). All pastes created with Configuration 1 wereproduced using 0.6M Na-GA solution at a liquid weight to powder weightratio (L/P) value of 0.23.

Twenty-five (25) 15 cc injectable kits of a modified SR material(Configuration 2 comprising 1.290 wt % of <45 μm Na-GA powder) wereprepared (35.00 g±0.01 g powder and 7.59 g±0.01 g sterile water forirrigation) from a 1013.071 g batch blended for 20 min in a 2 qtstainless V-shell using a 60 Hz P-K Twin-Shell Yoke blender. The waterwas overfilled by 0.10 g to account for solution loss in the vial duringtransfer. The kits were subjected to gamma radiation sterilization(25-32 kGy dosage). Four of these kits were used for this study.

The L/P value for Configuration 2 is 0.214. The difference in L/P valuesfor the two configurations is due to the movement of the Na-GA from thesolution to the powder.

Results:

The results of the Vicat set time showed Configuration 2 to have shiftedthe Vicat set time out by a small amount. Other than the location of theNa-GA within the two configurations, the only other variable is that theConfiguration 2 kits were irradiated, while the Configuration 1materials were not. To address these two variables, Vicat set time oftwo additional samples for each configuration were taken; however, theConfiguration 2 samples were not subjected to sterilization.

Two 35 g units of Configuration 1 powder were tested for Vicat set time.The entire mix was transferred to a 50 mL polystyrene beaker cup (VWRScientific P/N 13916-015); the paste was leveled and major air pocketswere removed through the gentle tapping of the cup on a table. Vicat settime was determined through the same method as performed above on bothsamples.

Two units of Configuration 2 powder were tested, and the entire mix wasused to determine Vicat set times as performed in the previousparagraph. One of the mixes was performed with 30 g of powder due tolack of material.

The new data obtained for Configuration 1 was combined with results fromthe previous Vicat testing since there was no difference in thetreatment of the specimens other than volume. The new data forConfiguration 2 was used independently to compare against theConfiguration 1 results.

FIG. 5 shows the overlaid curves of the titrations of the 1M G solutionsproduced from crystalline GA with and without gamma sterilization. Theresulting curves are indistinguishable. As noted in Example 3, solutionsof Na-GA used in the manufacturing of Configuration 1 kits displayed apH shift post gamma radiation sterilization. However, this change in pHwas not seen for a solution created with GA gamma irradiated in thecrystalline form. This result is indicative that degradation via gammairradiation of the glycolate ion is greatly, if not completely,alleviated by exposure in the crystalline form. This is strong evidencethat the crystalline Na-GA component in Configuration 2 will also beless affected by gamma irradiation.

Table 2 below shows the average results from the 24 hr dry DTS testingof each configuration. Both configurations exhibited DTS values close to9 MPa with less than a 10% coefficient of variance within each group.Although Configuration 2 exhibited a slightly higher average strengthvalue of 9.29 MPa, the difference between the two configurations was notstatistically significant (p=0.25). The observed difference can beattributed to error inherent of the test methodology. These results showthat the final set cement of both configurations exhibit the samemechanical strengths. TABLE 2 24 hr DTS (MPa), n = 6 Avg. Configuration[SD] 1 8.80 [0.62] 2 9.29 [0.75]

Table 3 below shows the average results from the four day dissolutiontest of each configuration. The two configurations exhibited almostidentical dissolution results with average weight percent remainingvalues of 63% after four days. The similarity in the measurements shownfor each configuration is further justification of both systemsresulting in the same reaction chemistries and extent of reactions.TABLE 3 4 Day Dissolution (wt % remaining), n = 5 Configuration Avg.[SD] 1 63.24 [3.72] 2 62.55 [1.94]

SEM micrographs of typical features seen throughout the bulk of the setcements taken of the fracture surface of a DTS specimen made from eachof the configurations were reviewed. The final products of eachconfiguration are substantially identical based on this microscopicevaluation.

Table 4 below shows the average injection force and Vicat set timeresults for each configuration. Both configurations exhibit very similarinjection force results with averages differing by less than 10 N, whichis less than 3% of the overall average. The coefficients of variance forboth measurements are below 6%, demonstrating good reproducibility inthe methodology. The average injection force of Configuration 2, 336.9N, was slightly (2.6%) lower than that of Configuration 1. These resultsshow equivalence in the viscosities and flow characteristics of pastesmade from both configurations. TABLE 4 Injection Force and Vicat SetTime, n = 3 Avg. Inj. Force (N) Avg. Set Time Configuration [SD] (mm:ss)[SD] 1 346.0 [19.6] 15:00 [00:30] 2 336.9 [13.4] 17:40 [01:26]

The two configurations did exhibit a difference in the Vicat set timemeasurements. The average Vicat set time for Configuration 2 was 17:40(mm:ss), which is 2:40 longer than that seen for Configuration 1. With astandard deviation of 30 sec, the Configuration 1 measurements resultedin a very tight data spread in comparison to the Configuration 2 data,the standard deviation of which was 1:26. There is clearly a differencebetween the Vicat set time of Configuration 1 and gamma sterilizedConfiguration 2.

In order to address the shift in Vicat set time shift seen for theirradiated Configuration 2 kits, two additional Vicat set timemeasurements were taken of each configuration. The Configuration 2powder retained prior to sterilization was used to determine if theshift was induced by the radiation or from the relocation of the Na-GA.Table 5 below shows the average Vicat set time results for the twoconfigurations. Results presented for Configuration 1 are the combinedresults from the two additional units as well as the three measurementspresented above. TABLE 5 Vicat Set Time for Unsterilized Configurations,n = 5 Config. 1; n = 2 Config. 2 Configuration Avg. Set Time (mm:ss)[SD] 1 14:18 [01:02] 2 14:45 [00:21]

In this scenario, the Vicat set times of each configuration matched upvery nicely with the difference between the averages being under 30 s,unlike what was seen above for the irradiated Configuration 2 data. Thisshows that the reaction kinetics for the two configurations result invery similar Vicat set times, and further demonstrates equivalencebetween the two configurations. The shift in Vicat set time seen in thedata presented earlier was the result of gamma irradiation and notdifferences between the two configurations.

The observation that gamma irradiation induces a shift of Vicat set forConfiguration 2 was not unexpected. This observation is consistent withExample 3, where a Configuration 1 type blend of powder showed anincreasing average Vicat set time with consecutive doses of gammairradiation of the Na-GA solution, in the same dose range.

Conclusion:

No statistically significant differences between DTS, dissolution, andinjection force values were observed between the two productconfigurations. A statistical difference in the Vicat set time valueswas observed when the irradiated Configuration 2 data was evaluated(p-value=0.04), but no difference was seen when the analysis wasperformed with the unsterilized Configuration 2 data (p-value=0.59).This difference can not be blamed on the configuration changes as thesecond Vicat set time comparison would have resulted in a significantdifference as well if relocation of the Na-GA was the cause. Thus, thisstudy shows chemical, physical, mechanical, and morphologicalequivalence between the two configurations in both the paste and setcement forms.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

1. A method for improving the storage stability of a kit comprising a particulate composition and an aqueous solution adapted for forming a bone graft substitute cement upon mixing, wherein the kit includes calcium phosphate powders reactive to form brushite in the presence of water and a carboxylic acid, the method comprising: i) packaging a monocalcium phosphate monohydrate powder and a β-tricalcium phosphate powder in separate containers in the kit; and ii) packaging the carboxylic acid in the kit either in the form of a crystalline powder or dissolved in the aqueous solution, with the proviso that when the carboxylic acid is dissolved in the aqueous solution, it is added to the solution after radiation sterilization of the aqueous solution.
 2. The method of claim 1, wherein the kit further comprises calcium sulfate hemihydrate powder, and the method further comprises packaging the calcium sulfate hemihydrate powder in a separate container, or in admixture with one or both of the monocalcium phosphate monohydrate powder and the β-tricalcium phosphate powder.
 3. The method of claim 1, wherein the carboxylic acid is in the form of a neutralized salt.
 4. The method of claim 3, wherein the neutralized salt is an alkali metal salt.
 5. The method of claim 3, wherein the neutralized salt is selected from the group consisting of sodium glycolate, potassium glycolate, sodium lactate, and potassium lactate.
 6. The method of claim 1, further comprising irradiating the components of the kit with gamma radiation for sterilization.
 7. The method of claim 1, wherein the carboxylic acid crystalline powder is packaged separately in a container.
 8. The method of claim 1, wherein the carboxylic acid crystalline powder is packaged in the container containing the monocalcium phosphate monohydrate powder or in the container containing the β-tricalcium phosphate powder.
 9. A bone graft substitute kit, comprising: i) a first container enclosing a monocalcium phosphate monohydrate powder; ii) a second container enclosing a β-tricalcium phosphate powder; iii) a calcium sulfate hemihydrate powder enclosed within a separate container or admixed with one or both of the monocalcium phosphate monohydrate powder and the β-tricalcium phosphate powder; iv) an aqueous solution enclosed within a separate container; and v) a carboxylic acid dissolved within the aqueous solution or present in the form of a crystalline powder, the carboxylic acid crystalline powder being enclosed within a separate container or admixed with any one or more of the monocalcium phosphate monohydrate powder, the β-tricalcium phosphate powder, and the calcium sulfate hemihydrate powder, with the proviso that when the carboxylic acid is dissolved in the aqueous solution, it is added to the solution after radiation sterilization of the aqueous solution.
 10. The bone graft substitute kit of claim 9, further comprising a written instruction set describing a method of using the kit.
 11. The bone graft substitute kit of claim 9, wherein the kit is sterilized by exposure to gamma radiation.
 12. The bone graft substitute kit of claim 1, wherein the carboxylic acid is in the form of a neutralized salt.
 13. The bone graft substitute kit of claim 12, wherein the neutralized salt is an alkali metal salt.
 14. The bone graft substitute kit of claim 12, wherein the neutralized salt is selected from the group consisting of sodium glycolate, potassium glycolate, sodium lactate, and potassium lactate.
 15. The bone graft substitute kit of claim 9, wherein the calcium sulfate hemihydrate powder is enclosed within a separate container.
 16. The bone graft substitute kit of claim 9, wherein the carboxylic acid crystalline powder is admixed with any one or more of the monocalcium phosphate monohydrate powder, the β-tricalcium phosphate powder, and the calcium sulfate hemihydrate powder.
 17. The bone graft substitute kit of claim 9, wherein the carboxylic acid crystalline powder is enclosed within a separate container such that the carboxylic acid crystalline powder can be reconstituted by admixture with the aqueous solution prior to mixing the aqueous solution with one or more of the monocalcium phosphate monohydrate powder, the β-tricalcium phosphate powder, and the calcium sulfate hemihydrate powder.
 18. The bone graft substitute kit of claim 9, wherein the calcium sulfate hemihydrate powder further includes, in admixture, an accelerant adapted for accelerating the conversion of calcium sulfate hemihydrate to calcium sulfate dihydrate.
 19. The bone graft substitute kit of claim 18, wherein the accelerant is selected from the group consisting of calcium sulfate dihydrate particles, potassium sulfate particles, and sodium sulfate particles, wherein the accelerant is optionally coated with sucrose.
 20. The bone graft substitute kit of claim 9, further comprising β-tricalcium phosphate granules in a separate container or in admixture with one or more of the monocalcium phosphate monohydrate powder, the β-tricalcium phosphate powder, and the calcium sulfate hemihydrate powder.
 21. The bone graft substitute kit of claim 9, wherein the calcium sulfate hemihydrate is α-calcium sulfate hemihydrate.
 22. The bone graft substitute kit of claim 9, wherein the calcium sulfate hemihydrate powder has a bimodal particle distribution comprising about 30 to about 60 volume percent of particles having a mode of about 1.0 to about 3.0 microns and about 40 to about 70 volume percent of particles having a mode of about 20 to about 30 microns, based on the total volume of the calcium sulfate hemihydrate powder.
 23. The bone graft substitute kit of claim 9, wherein the β-tricalcium phosphate powder has a bimodal particle size distribution comprising about 30 to about 70 volume percent of particles having a mode of about 2.0 to about 6.0 microns and about 30 to about 70 volume percent of particles having a mode of about 40 to about 70 microns based on the total volume of the β-tricalcium phosphate powder.
 24. The bone graft substitute kit of claim 23, wherein the β-tricalcium phosphate powder has a bimodal particle size distribution comprising about 50 to about 65 volume percent of particles having a mode of about 4.0 to about 5.5 microns and about 35 to about 50 volume percent of particles having a mode of about 60 to about 70 microns based on the total volume of the β-tricalcium phosphate powder.
 25. The bone graft substitute kit of claim 9, further comprising a biologically active agent enclosed within a separate container or admixed with any one or more of the monocalcium phosphate monohydrate powder, the β-tricalcium phosphate powder, and the calcium sulfate hemihydrate powder.
 26. The bone graft substitute kit of claim 25, wherein the biologically active agent is selected from the group consisting of cancellous bone chips, growth factors, antibiotics, pesticides, chemotherapeutic agents, antivirals, analgesics, and anti-inflammatory agents.
 27. The bone graft substitute kit of claim 25, wherein the biologically active agent is an osteoinductive material.
 28. The bone graft substitute kit of claim 27, wherein the osteoinductive material is demineralized bone matrix.
 29. The bone graft substitute kit of claim 26, wherein the biologically active agent is a growth factor selected from the group consisting of fibroblast growth factors, platelet-derived growth factors, bone morphogenic proteins, osteogenic proteins, transforming growth factors, LIM mineralization proteins, osteoid-inducing factors, angiogenins, endothelins; growth differentiation factors, ADMP-1, endothelins, hepatocyte growth factor and keratinocyte growth factor, heparin-binding growth factors, hedgehog proteins, interleukins, colony-stimulating factors, epithelial growth factors, insulin-like growth factors, cytokines, osteopontin, and osteonectin.
 30. A bone graft substitute kit, comprising: i) a first container enclosing a monocalcium phosphate monohydrate powder; ii) a second container enclosing a β-tricalcium phosphate powder having a median particle size of less than about 20 microns; iii) an α-calcium sulfate hemihydrate powder enclosed within a separate container or admixed with the β-tricalcium phosphate powder in the second container, the α-calcium sulfate hemihydrate powder having a bimodal particle distribution and a median particle size of about 5 to about 20 microns; iv) an aqueous solution enclosed within a separate container; v) a carboxylic acid in the form of a crystalline powder, the carboxylic acid crystalline powder being enclosed within a separate container, wherein the carboxylic acid is in the form of a neutralized alkali metal salt; vi) an accelerant adapted for accelerating the conversion of calcium sulfate hemihydrate to calcium sulfate dihydrate in admixture with the α-calcium sulfate hemihydrate powder; and vii) β-tricalcium phosphate granules in a separate container or in admixture with one or both of the β-tricalcium phosphate powder and the calcium sulfate hemihydrate powder, wherein the granules have a median particle size of at least about 75 microns. 